Culture Conditions and Preparation of Squashes

The best polytene chromosomes spreads are produced from late, third instar larvae that have been well fed with yeast and grown in uncrowded cultures.

Culture media can be any one of the three mentioned in Subheading 2.

1. For larger chromosomes, culture second and third instar larvae at 18°C and feed additional yeast (see Note 1.)

2. Using a dissecting needle, select large, third instar larvae that have migrated from the medium but are not about to pupate or have everted their spiracles. Place larvae in a container (Petri dish, Syracuse watch glass) that has a thin layer of PBS, Drosophila Ringers, or 0.8% saline and shake the container to rinse away media adhering to the body wall.

3. Determine the sex of the larva if necessary by looking for the gonadal area (see Fig. 1).

4. Transfer a single larva using forceps to a glass slide containing a large drop of Drosophila Ringers or 45% acetic acid (see Note 2) and place the slide on the stage of a dissecting scope with transmitted light (see Note 3).

5. Firmly grasp the head end of the larva at the base of the mouth hooks with a pair of forceps and quickly grasp the tail end about three-quarters along the length of the body with a second pair of forceps. Pull off the head end (see Note 4).

6. Remove the glands (see Fig. 2) as quickly as possible and place in a second drop of 45% acetic acid. Clean off fatty tissue, although the small thin strips along the length of the gland can remain without affecting the preparation Also remove the narrow ducts at the anterior end of the glands.

7. Allow glands to sit in 45% acetic acid fix for 2-5 min.

8. Remove the glands to a drop of lactic-acetic-orcein stain on a slide and allow to stain for 5 min. Do not let stain evaporate.

9. Prepare a clean slide for the squash by wiping with a Kimwipe. No lint should be seen on the slide (see Note 5).

Drosophila Larvae Slide

Fig. 1. Male and female larvae with gonadal area visible through body wall as indicated by arrow. Male exhibits a large oval translucent area, whereas the female exhibits a very small translucent spot, sometimes difficult to locate. (Dissecting microscope at 25x; reduced from original magnification.)

Fig. 1. Male and female larvae with gonadal area visible through body wall as indicated by arrow. Male exhibits a large oval translucent area, whereas the female exhibits a very small translucent spot, sometimes difficult to locate. (Dissecting microscope at 25x; reduced from original magnification.)

10. Add a small drop (20 |L) of 1 : 2 : 3 fixative to the slide and transfer the two glands from the stain to the drop (see Note 6).

11. Drop a clean cover slip on the preparation, allow fixative to spread, and gently tap the cover slip with a dissecting needle or blunt pencil while observing the preparation under the dissecting scope.

12. Holding a piece of bibulous paper along one edge of the cover slip, gently score the surface of the cover slip in a spiral or zigzag pattern to spread the chromosomes. Be careful not to shift the cover slip, as the motion will shear the chromosomes into fragments.

13. Blot the squash by pressing down with your thumb on a piece of bibulous paper overlaying the cover slip. Again, be careful not to allow the cover slip to shift.

14. Seal the edges of the cover slip with nail polish. For a permanent mount, see Note 7.

15. Store slides in the refrigerator for the short term or in the freezer for the long term.

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